Effect of leucine supplementation on indices of muscle damage following drop jumps and resistance exercise

The purpose of this study was to determine the effect of leucine supplementation on indices of muscle damage following eccentric-based resistance exercise. In vitro, the amino acid leucine has been shown to reduce proteolysis and stimulate protein synthesis. Twenty-seven untrained males (height 178.6 ± 5.5 cm; body mass 77.7 ± 13.5 kg; age 21.3 ± 1.6 years) were randomly divided into three groups; leucine (L) (n = 10), placebo (P) (n = 9) and control (C) (n = 8). The two experimental groups (L and P) performed 100 depth jumps from 60 cm and six sets of ten repetitions of eccentric-only leg presses. Either leucine (250 mg/kg bm) or placebo was ingested 30 min before, during and immediately post-exercise and the morning of each recovery day following exercise. Muscle function was determined by peak force during an isometric squat and by jump height during a static jump at pre-exercise (PRE) and 24, 48, 72, and 96 h post-exercise (24, 48, 72, 96 h). Additionally, at these time points each group’s serum levels of creatine kinase (CK) and myoglobin (Mb) along with perceived feelings of muscle soreness were determined. None of the C group dependent variables was altered by the recurring testing procedures. Peak force was significantly decreased across all time points for both experimental groups. The L group experienced an attenuated drop in mean peak force across all post-exercise time points compared to the P group. Jump height significantly decreased from PRE for both the L and P group at 24 h and 48 h. CK and Mb was significantly elevated from PRE for both experimental groups at 24 h. Muscle soreness increased across all time points for the both the L and P group, and the L group experienced a significantly higher increase in mean muscle soreness post-exercise. Following exercise-induced muscle damage, high-dose leucine supplementation may help maintain force output during isometric contractions, however, not force output required for complex physical tasks thereby possibly limiting its ergogenic effectiveness.     

Conformational remodeling of proteasomal substrates by PA700, the 19 S regulatory complex of the 26 S proteasome

PA700, the 19 S regulatory complex of the 26 S proteasome, plays a central role in the recognition and efficient degradation of misfolded proteins. PA700 promotes degradation by recruiting proteasomal substrates utilizing polyubiquitin chains and chaperone-like binding activities and by opening the access to the core of the 20 S proteasome to promote degradation. Here we provide evidence that PA700 in addition to binding misfolded protein substrates also acts to remodel their conformation prior to proteolysis. Scrambled RNase A (scRNase A), a misfolded protein, only slowly refolds spontaneously into an active form because of the rate-limiting unfolding of misfolded disulfide isomers. Notably, PA700 accelerates the rate of reactivation of scRNase A, consistent with its ability to increase the exposure of these disulfide bonds to the solvent. In this regard, PA700 also exposes otherwise buried sites to digestion by exogenous chymotrypsin in a polyubiquitinated enzymatically active substrate, pentaubiquitinated dihydrofolate reductase, Ub(5)DHFR. The dihydrofolate reductase ligand methotrexate counters the ability of PA700 to promote digestion by chymotrypsin. Together, these results indicate that in addition to increasing substrate affinity and opening the access channel to the catalytic sites, PA700 activates proteasomal degradation by remodeling the conformation of protein substrates.     

Distinct Functional Properties of Isoamylase-Type Starch Debranching Enzymes in Monocot and Dicot Leaves

Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.Semicrystalline starch enables photosynthetic eukaryotes to store large quantities of Glc over extended time periods compared with other species, in which the soluble polymer glycogen functions to store carbohydrate reserves (Ball and Morell, 2003). Eukaryotes gained the capacity to photosynthesize after the capture of a cyanobacterial endosymbiont by a glycogen-metabolizing host cell. In the lineage that evolved subsequently, known as the Archaeplastida, select glucan-storage enzymes encoded within the host nucleus, the endosymbiont, and potentially a prokaryotic parasite located within the host cell developed so as to generate the branched glucan polymer amylopectin (Ball et al., 2011, 2013). Such molecules are highly similar to glycogen in terms of chemical structure, but the molecular architecture of amylopectin enables the formation of semicrystalline structures (Buléon et al., 1998). These latter then assemble into higher order structures leading to starch granule formation. The advent of starch granules is likely to have been critical for the evolution of chloroplast-containing organisms, including the spread of land plants on the Earth’s surface, because they enable the storage of photosynthetically generated Glc for many hours in tissues such as leaves during diurnal cycles or for months to years in seeds.An important aspect of the evolutionary change from glycogen to starch is the use of particular α(1→6)-glucosidases, referred to as isoamylase-type starch debranching enzymes (ISA), in the production of amylopectin (Ball et al., 1996; Myers et al., 2000; Hennen-Bierwagen et al., 2012). A suite of genes encoding the enzymes that accomplish starch biosynthesis was established early in the evolution of chloroplast-containing organisms (i.e. the Chloroplastida) prior to the divergence of distantly related groups including green algae and land plants. Included in this gene set are three paralogs that encode the proteins ISA1, ISA2, and ISA3, each of which is highly conserved in chloroplast-containing species. ISA1 of vascular plants and bryophytes, for example, are approximately 70% identical over more than 600 residues, and between land plants and prasinophyte algae this value is about 60%. ISA1 or ISA2 deficiencies in potato (Solanum tuberosum) tuber, Arabidopsis (Arabidopsis thaliana) leaf, Chlamydomonas reinhardtii cells, and cereal endosperms result in reduced starch content, altered amylopectin structure, and the appearance of soluble, branched glucans similar to native glycogen (James et al., 1995; Mouille et al., 1996; Nakamura et al., 1996; Bustos et al., 2004; Delatte et al., 2005; Wattebled et al., 2005). Such soluble polymers, referred to as phytoglycogen, have not been observed in wild-type plants. Thus, ISA1 and ISA2 functions are important determinants of whether storage glucans are semicrystalline or soluble. ISA3, in contrast, functions primarily in starch catabolism (Wattebled et al., 2005; Delatte et al., 2006).ISA1 and ISA2 appear to function together in Arabidopsis leaf as a single entity, because essentially identical phenotypes are observed in single mutants lacking either protein or double mutants lacking both of them (Zeeman et al., 1998; Delatte et al., 2005; Wattebled et al., 2005). Biochemical analysis of native and recombinant proteins has shown directly that ISA1 and ISA2 function together in a complex. ISA activity was first purified from potato tuber and found to contain two distinct polypeptides identified as ISA1 and ISA2 (Ishizaki et al., 1983; Hussain et al., 2003). Heteromultimers containing these two proteins were also purified from rice (Oryza sativa) and maize (Zea mays) endosperm (Utsumi and Nakamura, 2006; Kubo et al., 2010). Finally, a mixture of native and recombinant rice proteins demonstrated directly that specific enzymatic activities are provided by ISA1 and ISA2 functioning together in a heteromultimeric complex (Utsumi and Nakamura, 2006). ISA1 is the catalytic subunit within this complex, whereas ISA2 is noncatalytic, owing to amino acid substitutions at residues that are essentially invariant in the GH13 family of glycoside hydrolases (i.e. the α-amylase superfamily), several of which participate in the catalytic mechanism (Hussain et al., 2003; Utsumi and Nakamura, 2006). Despite lacking catalytic activity, ISA2 proteins are conserved in all chloroplast-containing species that have been examined, which rules out recently evolved mutations and, to the contrary, suggests a functional selective advantage.The necessity for the ISA1/ISA2 heteromultimer is not obvious in light of the fact that, in some instances, ISA1 by itself can condition normal levels of starch and the suppression of phytoglycogen accumulation. Cyanidioschyzon merolae, a species within the Rhodophyta lineage of the Archaeplastida family, contains semicrystalline starch and amylopectin with physical characteristics similar to that of Chloroplastida species (Hirabaru et al., 2010). The C. merolae genome contains elements that encode ISA1 and ISA3 yet lacks a homolog encoding ISA2 (Coppin et al., 2005). Thus, in some instances, starch can be generated, and phytoglycogen accumulation suppressed, without an ISA2 protein. Cereal endosperms provide additional evidence that ISA2 is not strictly required for normal starch levels and the suppression of phytoglycogen accumulation. Mutants or transgenic lines lacking ISA2 are known in rice (Utsumi et al., 2011) and maize (Kubo et al., 2010). Endosperm from these plants exhibits normal starch levels, with amylopectin structure essentially the same as the wild type, and lacks phytoglycogen. ISA activity presumably is provided in the endosperm of these mutants by a homomultimeric enzyme containing only ISA1.The reason why ISA2 is strictly conserved in the Chloroplastida is not understood yet. Two explanations can be considered. One possibility is that the inherent structure of ISA1 in cereals, resulting from mutations accumulated specifically in this evolutionary lineage, allows it to act without ISA2. Another possibility is that metabolic differences in specific tissues (e.g. leaf versus endosperm) require specialized enzymatic properties of the ISA1/ISA2 heteromer that ISA1 by itself does not provide. To test these hypotheses, this study combined maize and Arabidopsis ISA1 and ISA2 isoforms both in vitro and in vivo. Maize ISA1 was found to be active without any ISA2 protein, either in vitro or in Arabidopsis leaves, whereas Arabidopsis ISA1 required an ISA2 partner in all instances. Thus, ISA1 appears to have evolved in the cereal lineage so that it no longer requires ISA2 for enzymatic activity or metabolic function in the generation of starch and the suppression of phytoglycogen accumulation.     

Murine expression and mutation analyses of the prostate androgen-regulated mucin-like protein 1 (Parm1) gene, a candidate for human epispadias

Background

Epispadias is the mildest phenotype of the human bladder exstrophy–epispadias complex (BEEC), and presents with varying degrees of severity. This urogenital birth defect results from a disturbance in the septation process, during which separate urogenital and anorectal components are formed through division of the cloaca. This process is reported to be influenced by androgen signaling. The human PARM1 gene encodes the prostate androgen-regulated mucin-like protein 1, which is expressed in heart, kidney, and placenta.

Methods

We performed whole mount in situ hybridization analysis of Parm1 expression in mouse embryos between gestational days (GD) 9.5 and 12.5, which are equivalent to human gestational weeks 4–6. Since the spatio-temporal localization of Parm1 corresponded to tissues which are affected in human epispadias, we sequenced PARM1 in 24 affected patients.

Results

We found Parm1 specifically expressed in the region of the developing cloaca, the umbilical cord, bladder anlage, and the urethral component of the genital tubercle. Additionally, Parm1 expression was detected in the muscle progenitor cells of the somites and head mesenchyme. PARM1 gene analysis revealed no alterations in the coding region of any of the investigated patients.

Conclusions

These findings suggest that PARM1 does not play a major role in the development of human epispadias. However, we cannot rule out the possibility that a larger sample size would enable detection of rare mutations in this gene.     

A top-down proteomics approach for differentiating thermal resistant strains of Enterobacter sakazakii

Thermal tolerance has been identified as an important factor relevant to the pathogenicity of Enterobacter sakazakii in human neonates. To identify a biomarker specific for this phenotypic trait, intact protein expression profiles of 12 strains of E. sakazakii were obtained using liquid chromatography mass spectrometry. Proteins were extracted from the bacterial cells, separated by reversed-phase liquid chromatography and mass analyzed. At the end of the chromatography run, the uncharged masses of the multiply charged proteins were determined via automated software routines. The resulting data provided an accurate mass expression profile of the proteins found in the individual strains. From the individual expression profiles, it was possible to identify unique proteins corresponding to strains with thermal resistance. One protein found only in the thermal tolerant strains was sequenced and identified as homologous to a hypothetical protein found in the thermal tolerant bacteria, Methylobacillus flagellatus KT. The protein sequence of this protein was then used to reverse-engineer PCR primers for the gene sequence associated with the protein. In all cases, only thermal tolerant strains of E. sakazakii produced amplified PCR products, demonstrating the specificity of this biomarker.     

Cholesterol-induced macrophage apoptosis requires ER stress pathways and engagement of the type A scavenger receptor

Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)-induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.